PROJECT SUMMARY Both neurodevelopment and the synaptic plasticity events that underlie learning and memory rely heavily on tightly regulated gene expression programs and rapid, finely-tuned translation of messenger RNA (mRNA) transcripts. The degradation, stability, and translation of mRNA has, in recent years, been found to be regulated by adenosine methylation, which alters both transcript structure and the recruitment of RNA-binding proteins that inform these activities. Recent identification of and experimentation with the methyltransferases (?writers?), demethylases (?erasers?), and specific methyladenosine binding proteins (?readers?) have established that these epitranscriptomic mRNA regulatory processes are both dynamic and tightly regulated. Although the most well-studied of these modifications is N6-methyladenosine (m6A), N1- methyladenosine (m1A) has also recently emerged as a prevalent epitranscriptomic mark. Current methods used to explore these modifications require large sample sizes and are inherently low-resolution. These limitations preclude them from mapping and quantifying the epitranscriptome in specific brain regions, or in clinical biospecimens. Here, we describe preliminary development of innovative technologies to precisely sequence and probe the function of specific m6A and m1A modifications. We propose to leverage these foundations in the service of the following specific aims: 1) Evolve and establish high-resolution, antibody-free m6A and m1A mapping platforms for brain analysis, 2) Design and validate a molecular toolkit to manipulate transcript-specific m6A and m1A modifications in vivo, and 3) Catalog m6A and m1A modifications in the brain across development, neuron populations, activity state, and in synapses, and determine their function in relation to learning and memory. Our findings will illuminate how the epitranscriptomic landscape and specific mRNA transcripts in discrete neuronal populations regulates gene expression to inform complex neuronal processes, such as development, learning and memory, and how perturbations thereof result in abnormal brain function such as learning impairment. Importantly, this translational, functional validation of our new tools, which will be made available to the research community, provides a strong foundation for their usage to specifically interrogate how mRNA modifications are perturbed in other pathological contexts.